PROJECT SUMMARY Past influenza cross-species transmission events have lead to devastating human pandemics. H5N1 is an avian influenza virus that has caused recurrent, high pathogenicity human infections since 1997. Humans usually acquire H5N1 through interaction with live birds, and mounting evidence suggests that H5N1 circulation in poultry is strongly linked to human infection. Despite this, the genetic and environmental factors that promote H5N1 circulation in poultry remain unknown. A predominant hypothesis is that wild birds seed new viruses into poultry, and humans acquire infection via poultry interaction. However, the rate of transmission between wild birds and poultry has never been estimated. Although certain husbandry practices like outdoor rearing and transport to large, live poultry markets are hypothesized to enhance H5N1 circulation, the relative contributions of these husbandry practices have never been systematically assessed. Finally, virologic studies have produced a catalogue of mutations associated with human adaptation in laboratory and animal studies, which are currently used to query emerging H5N1 strains and assess pandemic risk. However, many human-infecting H5N1 strains lack known markers of adaptation, and it is unclear whether these mutations predict spillover risk in nature. In this proposal, I will use phylogenetic and statistical methods to determine the genetic and environmental drivers of H5N1 cross-species transmission through 3 specific aims. Completion of these projects with my mentors and co-mentors will allow me to achieve my career goal of transitioning to an independent faculty role by the end of the K99 phase. 1. I will use a recently developed structured coalescent model to estimate the rate of H5N1 transmission between wild birds, poultry, and humans. I hypothesize that cross-species transmission occurs frequently between wild birds and poultry, but only a small subset of lineages circulate long-term. I expect to observe ongoing transmission in poultry, but not in humans. 2. I will use phylogenetic and statistical methods to determine the environmental and husbandry practices that promote long-term H5N1 circulation in poultry. I hypothesize that short-term spillover events will be associated with outdoor poultry housing and rice cropping. Long-term establishments will be correlated with poor vaccination coverage and introduction into a large poultry market. 3. Elucidate genetic and phenotypic determinants of cross-species transmission. I will combine the power of a genome-wide scan with phenotypic validation to identify the genetic correlates of avian influenza spillover. I hypothesize that H5N1 lineages that are prone to human spillover will be enriched for mutations experimentally linked to host switching. I predict that our scan will identify mutations that elicit improved human receptor binding, enhanced replication in mammalian cells, and abrogation of interferon production.